Skin, the largest human organ, plays an important part in the body's defense against invasion by infectious agents and contact with noxious substances. But this barrier function of the skin appears to have prevented the art from appreciating that transcutaneous immunization provided an effective alternative to enteral, mucosal, and parenteral administration of vaccines.
Anatomically, skin is composed of three layers: the epidermis, the dermis, and subcutaneous fat. Epidermis is composed of the basal, the spinous, the granular, and the cornified layers; the stratum corneum comprises the cornified layer and lipid. The principal antigen presenting cells of the skin, Langerhans cells, are reported to be in the mid- to upper-spinous layers of the epidermis in humans. Dermis contains primarily connective tissue. Blood and lymphatic vessels are confined to the dermis and subcutaneous fat.
The stratum corneum, a layer of dead skin cells and lipids, has traditionally been viewed as a barrier to the hostile world, excluding organisms and noxious substances from the viable cells below the stratum corneum. Stratum corneum also serves as a barrier to the loss of moisture from the skin: the relatively dry stratum corneum is reported to have 5% to 15% water content while deeper epidermal and dermal layers are relatively well hydrated with 85% to 90% water content. Only recently has the secondary protection provided by antigen presenting cells (e.g., Langerhans cells) been recognized. Moreover, the ability to immunize through the skin with or without penetration enhancement (i.e., transcutaneous immunization) using a skin-active adjuvant has only been recently described. Although undesirable skin reactions such as atopy and dermatitis were known in the art, recognition of the therapeutic advantages of transcutaneous immunization (TCl) might not have been appreciated in the past because the skin was believed to provide a barrier to the passage of molecules larger than about 500 daltons.
We have shown that a variety of adjuvants are effectively administered by TCl to elicit systemic and regional antigen-specific immune responses to a separate, co-administered antigen. See WO 98/20734, WO 99/43350, and WO 00/61184; U.S. Pat. Nos. 5,910,306 and 5,980,898; and U.S. patent application Ser. Nos. 09/257,188; 09/309,881; 09/311,720; 09/316,069; 09/337,746; and 09/545,417. For example, adjuvants like ADP-ribosylating exotoxins are safe and effective when applied epicutaneously, in contrast to the disadvantages associated with their use when administered by an enteral, mucosal, or parenteral route.
U.S. Pat. Nos. 4,220,584 and 4,285,931 use E. coli heat-labile enterotoxin to immunize against E. coli-induced diarrhea. Rabbits were intramuscularly injected with the immunogen and Freund's adjuvant. Protection against challenge with toxin and neutralization of toxic effects on ileal loop activity was shown. U.S. Pat. No. 5,182,109 describes combining vaccine and toxin (e.g., E. coli heat-labile toxin) and administration in injectable, spray, or oral form. Neutralization was demonstrated with colostrum of immunized cows. Mutant versions of enterotoxin have also been described to retain immunogenicity and eliminate toxicity (e.g., U.S. Pat. Nos. 4,761,372 and 5,308,835).
Novel and inventive vaccine formulations, as well as processes for making and using them, are disclosed herein. In particular, TCl and the advantages derived therefrom in human vaccination to treat diarrheal disease are demonstrated. An important showing is that competition among different antigens in a multivalent vaccine was not an obstacle when administered by transcutaneous immunization. Other advantages of the invention are discussed below or would be apparent from the disclosure herein.